Microbial oil mixtures and uses thereof

ABSTRACT

The present invention relates to compositions including blends of microbial oils, methods of using such compositions, particularly as supplements for infant formula, and methods of increasing the amount of long chain polyunsaturated fatty acids in infant formula.

This application is a division of application Ser. No. 07/944,739, filedSep. 14 1993, now U.S. Pat. No. 5,374,657, which is a File WrapperContinuation of Ser. No. 07/645,457, filed Jan. 24, 1991, now abandoned.

This invention relates to blends or mixtures of polyunsaturated fattyacid-containing microbial oils and to uses thereof. In a specificpreferred embodiment, this invention concerns the use of such oils as anadditive or supplement for human diets, for example, as an additive toinfant formula.

It long has been known that long chain polyunsaturated fatty acids(PUFAs) are essential to the human diet, particularly during periods ofrapid tissue growth. Sanders et al, Am. J. Clin. Nutr., 31:805-813(1978). Certain of these long chain acids, such as arachidonic acid(ARA), cannot be synthesized de novo in humans. Only by metabolizinglinoleic acid (LOA), which is connected to gamma linolenic acid (GLA),and then to ARA can the human body produce ARA. LOA, in turn, is anessential acid which can only be obtained from dietary sources.Additionally, the presence of eicosapentaenoic acid (EPA) in the dietinhibits the metabolic conversion of LOA to ARA. Carlson, et al.,INFORM, 1:306 (1990). ARA and docosahexaneoic acid (DHA) are criticalelements of muscle, organ and vascular tissues.

Infancy is the most significant period of rapid growth in a human'slife. An infant can increase its body weight by three times or moreduring its first year of life. Accordingly, it is critical that theinfant receive adequate amounts of PUFAs to insure proper structural andorgan development. Human breast milk contains high levels of PUFAs inwhich the ratio of ARA to EPA is typically about 20:1. However, manywomen choose not to breast feed their infants for either part or all ofthe first year of the infant's life.

As recognized by Clandinin et al., U.S. Pat. No. 4,670,285, incorporatedherein by reference, available infant formulas are deficient in longchain (C₂₀ and C₂₂) PUFAs. Clandinin et al. disclose an infant formulaprepared from a blend of vegetable oil and egg yolk lipid and/or fishoil which can provide a total fat composition comparable to that ofhuman breast milk. A preferable composition comprises from 75 to 95parts by weight egg yolk and 5 to 25 parts vegetable oil. Thiscomposition is the entire lipid content of the infant formula and it isnot economical to prepare. Additionally, the infant formula disclosed byClandinin et al. results in an EPA level which is 16 times higher thanthe level of EPA in human breast milk and an ARA level which is only onequarter that of breast milk.

DE 3603000A1 (Milupa) discloses a computer profile of a highlypolyunsaturated acid fat mixture and discusses the use of such a mixtureto produce infant formulas. Sources of the fatty acids are listed ascertain types of macroalgae (i.e. seaweed), fish oil, organ fats frombeef and pork, and highly refined egg yolk oil. In addition to DHA fromfish oil, a potential source of DHA and ARA is said to be macroalgae,but only of the seaweed types. There is no suggestion to use microbes ofany type, much less microbial oil.

Methods of producing microbial oils are disclosed in the followingreferences, each of which is incorporated herein by reference. U.S. Pat.No. 5,244,912 discloses the production of eicosapentaneoicacid-containing single cell oils (EPASCO). U.S. Pat. No. 5,407,957discloses the production of docosahexaneoic acid-containing single celloil (DHASCO). Co-pending U.S. patent application Ser. No. 07/645,454,now abandoned filed concurrently with the present application andassigned to the same assignee, relates to the production of arachidonicacid-containing single cell oil (ARASCO). EP322,227 also discloses amicrobial oil production system. None these references teach the use ofblends containing unmodified microbial oils as a dietary supplement, orthe use of a blend of microbial oils as an additive to existing infantformula to provide that formula with a long chain PUFA compositionsimilar to breast milk.

Accordingly, it is an object of the present invention to provide aPUFA-enriched additive, the composition of which when added tocommercial infant formula will provide desired long chain PUFAs inamounts comparable to the amounts of those PUFAs found in human breastmilk.

It is an additional object of the present invention to provide aneconomical method of producing the above-described composition.

These, and other, objects are satisfied by the present invention asdescribed herein.

SUMMARY OF THE INVENTION

This invention relates to the use of microbial oils which contain longchain polyunsaturated fatty acids. Additionally, in various embodiments,fish oil and/or vegetable oils can be blended with such microbial oilsto form desired compositions. The compositions can be used as dietarysupplements, particularly as additives for infant formula, as well asfor pharmaceutical and cosmetic applications.

The invention also relates to economically viable processes for alteringthe long chain polyunsaturated fatty acid composition of infant formulaand/or baby food. Preferably, the altered composition resembles that ofhuman breast milk.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE INVENTION

Broadly stated, the present invention concerns blends, or mixtures,containing unmodified microbial oils. As used herein, "unmodified" meansnot chemically or covalently altered. It will be understood thatthroughout this specification references to "microbial oil" or "oil"mean, unless otherwise specifically stated, unmodified oil. "Microbialoils" or "single cell oils" are those oils naturally produced bymicroorganisms during their lifespan. Such oils can be high in longchain PUFAs. The applicant has discovered that certain of these oils,when blended with other microbial oils, fish oils, vegetable oils, orany combination thereof, can produce a composition useful for dietary,pharmaceutical or cosmetic purposes.

Various microbial oils, for example, can be obtained by, for example,the processes disclosed in above-referenced U.S. Pat. Nos. 5,244,921,5,407,957 EP322,227 (Yamada et al., Suntory) or U.S. patent applicationSer. No. 07/645,454, now abandoned (the latter having been filedconcurrently with the present application and assigned to the sameassignee). The disclosure of each of these references is specificallyincorporated by reference herein.

It is to be understood that the present invention encompasses the use ofa single-microbial oil containing at least two desirable PUFAs, such asARA and DHA. The oils specifically disclosed and utilized herein,however, each contain a single desirable PUFA.

Any non-toxic, PUFA-containing microbial oil can be used in the presentinvention. The most preferred microbial oils are those rich in anomega-3 or omega-6 PUFA, especially DHA, GLA or ARA. These PUFAstypically are missing from, or are inadequately provided in, dietarysupplements such as infant formulas or baby food. "Infant formula" asused herein means an enteral nutritional product which can besubstituted for human breast milk in feeding infants and typically iscomposed of a desired percentage of fat mixed with desired percentagesof carbohydrates and proteins in an aqueous solution. Frequentlymicronutrients, such as trace metals and vitamins or other desiredadditives are present. An exemplary formula is disclosed by Clandinin etal., U.S. Pat. No. 4,670,285, the disclosure of which is incorporatedherein by reference.

In the present invention, types of oils from different microbes can bemixed together to obtain a desired composition. Alternatively, oradditionally, PUFA-containing microbial oil can be blended with fishoil, vegetable oil or a mixture of both to obtain a desired composition.

An objective in mixing the oils is to obtain an additive which willprovide an infant formula with a desired omega-3 and omega-6 PUFAcomposition similar to that found in breast milk. While the proportionof the desired fatty acids in a microbial oil can vary, this proportioncan easily be determined and the amount of oil adjusted to provide thedesired amount of PUFA. Similarly, the percentage of desired PUFA infish oil or vegetable oils can easily be determined and the amount ofthe oil to be added can be adjusted as necessary to achieve the desiredresults.

"Fish oils" are those oils obtained from fish. Such oils typicallycontain DHA in amounts ranging from 3% to about 20%. Typically, however,fish oils also contain EPA which depresses the production of ARA in thebody. The addition of a microbial oil containing high levels of ARA tofish oil-containing compositions substantially overcomes that problem.

"Vegetable oil" includes all those oils from plants which contain longchain PUFAs. Typically, vegetable oils do not contain long chain PUFAs(PUFAs at least 20 carbons long), which is why animal organ oils areusually characterized as the source of PUFAs. Thus, vegetarians,especially vegetarian mothers, can have a diet containing inadequateamounts of PUFAs. Vegetable oils known to contain PUFAs may contain GLA.GLA is a C18:3 omega-6 PUFA. Such oils include black currant seed oil,borage oil and primrose oil. While GLA is the metabolic precursor toARA, the process of conversion is very slow, requiring the participationof the enzyme Δ6-desaturase. This enzyme is present in humans in verylow levels. Burre, et al., Lipids, 25:354-356 (1990). Thus, it would bepreferable to provide the body with ARA rather than its precursor, GLA.

Methods for isolating vegetable oils are known to those of skill in theart and do not comprise a part of the present invention. Additionally,certain fungi produce PUFA-containing oils. For example, Mucor speciesproduce a GLA-containing oil.

DHASCO, defined herein as docosahexaneoic acid-containing single celloil, can be obtained, for example, from Crypthecodinium cohnii asdisclosed in above-referenced U.S. Pat. No. 5,407,957. DHA is a C22:6omega-3 long chain PUFA.

EPASCO, defined herein as eicosapentaneoic acid-containing single celloil, can be obtained, for example, from Nitzschia alba as disclosed inabove-referenced U.S. Pat. No. 5,244,921. EPA is a C20:5 omega-3 longchain PUFA.

ARASCO, defined herein as arachidonic acid-containing single cell oil,can be obtained from species such as Pythium insidiosum, or Mortierellaalpina, as described in U.S. application Ser. No. 07/645,454 filedconcurrently herewith. ARA is a C20:4 omega-6 long chain PUFA.

Of those fungal species which previously have had their fatty acidscharacterized, it has been found that most do not make ARA. Weete, J.D., Fungal Lipid Biochemistry, Plenum Press, NY (1974). Of those specieswhich do make ARA, many, including all previously characterized Pythiumspecies, produce significant quantities of eicosapentaenoic acid (EPA)in addition to ARA. Table 1 sets forth the fatty acid profile of P.insidiosum as well as the fatty acid profile of other species of fungi.Unexpectedly, it has been found that P. insidiosum produces ARA withoutconcomitant production of EPA. As with fish oils, high EPA levels indietary supplements result in a depression of the ability to form ARAfrom dietary linoleic acid (LOA). Accordingly, while those fungalspecies producing both ARA and EPA can be utilized in the process ofthis invention, it is preferable to use species which do not producesignificant quantities of EPA. Such preferred species include Pythiuminsidiosum and Mortierella alpina. Both species are availablecommercially and are on deposit with the American Type CultureCollective in Rockville, Md., having accession numbers 28251 and 42430,respectively. Throughout this disclosure, unless otherwise expresslystated, P. insidiosum will be the representative fungal species.

One of the significant problems which an embodiment of the presentinvention overcomes, is the depression of ARA biosynthesis in infantscaused by the presence of enhanced dietary levels of EPA. This problemcan be corrected by providing ARA for use in infant formula at levelssubstantially similar to those found in human breast milk. Typically inhuman breast milk, the ratio of ARA:EPA is about 20:1 respectively. Thepresent invention specifically contemplates any microbial oil whichprovides a sufficient amount of ARA to overcome the negative effects ofdietary EPA. Preferably, the use of the ARA-containing oil will resultin an ARA:EPA ration of at least about 5:1. More preferably, the ratiowill be at least about 10:1 and, most preferably, it will be at leastabout 20:1. As can be seen, the higher the amount of ARA in the endproduct, with respect to the amount of EPA, the more desirable is theresult.

In a process of the present invention, the fungi are cultivated undersuitable ARA-containing oil producing cultivating conditions. Ingeneral, techniques of fungal cultivation are well known to those ofskill in the art and those techniques can be applied to the presentinventive process. For example, cultivation of an inoculating amount offungus can occur in submerged culture in shake flasks. The flask isprovided with a growth medium, seeded with fungal mycelium, and grown ona reciprocating shaker for about three to four days.

The composition of the growth medium can vary but always contains carbonand nitrogen sources. A preferred carbon source is glucose, amounts ofwhich can range from about 1. 0-100 grams glucose per liter of growthmedium. Typically about 15 grams/liter are utilized for shaker flaskculture. The amount can be varied depending upon the desired density ofthe final culture. Other carbon sources which can be used includemolasses, high fructose corn syrup, hydrolyzed starch or any other lowcost conventional carbon source used in fermentation processes.Additionally, lactose can be provided as a carbon source for P.insidiosum. Thus, whey permeate, which is high in lactose and is a verylow cost carbon source, can be used as a substrate. Suitable amounts ofthese carbon sources can readily be determined by those of skill in theart. Usually, additional carbon needs to be added during the course ofthe cultivation. This is because the organisms use so much carbon thatadding it all in a batch mode could prove unwieldy.

Nitrogen typically is provided in the from of yeast extract at aconcentration of from about 2 to about 15 grams extract per liter ofgrowth medium. Preferably, about four grams per liter are provided.Other nitrogen sources can be used, including peptone, tryptone,cornsteep liquor, etc. The amount to be added of these sources caneasily be determined by those of skill in the art. Nitrogen can be addedin a batch mode, i.e. all at one time prior to cultivation.

After cultivation for 3-4 days at a suitable temperature, typicallyabout 25°-30° C., an amount of fungi has grown which is sufficient foruse as an inoculum in a conventional stirred tank fermentor (STF). Suchfermentors are known to those of skill in the art and are commerciallyavailable. Fermentation can be carried out in batch, fed-batch, orcontinuous fermentation modes. Preferably, the STF is equipped with aRushton-type turbine impeller.

The fermentor is prepared by adding the desired carbon and nitrogensources. For example, a 1.5 liter fermentor can be prepared by mixingabout 50 grams of glucose and about 15 grams of yeast extract per literof tap water. As previously discussed, other carbon or nitrogen sourcesor mixtures thereof can be used.

The reactor containing the nutrient solution should be sterilized by,for example, heating prior to inoculation. After cooling to about 30°C., the inoculum can be added, and cultivation initiated. Gas exchangeis provided by air sparging. The air sparging rate can vary, butpreferably is adjusted to from about 0.5 to about 4.0 VVM (volume of airper volume of fermentor per minute). Preferably the dissolved oxygenlevel is kept at from about 10% to about 50% of the air saturation valueof the solution. Accordingly, adjustments in the sparge rate may berequired during cultivation. Agitation is desirable. The agitation isprovided by the impeller. Agitation tip speed preferably is set withinthe range of from about 50 cm/sec to about 500 cm/sec, preferably fromabout 100 to 200 cm/sec.

In general, the amount of inoculum can vary. Typically, from about 2% toabout 10% by volume of inoculum can be used. Preferably, in a fermentorseed train about 5% by volume of inoculum can be used.

Nutrient levels should be monitored. When glucose levels drop below 5g/l, additional glucose should be added. A typical cultivation cycleutilized about 100 grams of glucose and about 15 grams of yeast extractper liter. It is desirable to deplete the nitrogen during the course ofthe cultivation as this enhances oil production by the fungi. This isespecially true when M. alpina is used as the production organism.

Occasionally, the culture will produce an excessive quantity of foam.Optionally, an antifoaming agent, such as those known to those of skillin the art, e.g. Mazu 310®, can be added to prevent foam.

The temperature of cultivation can vary. However, those fungi whichproduce both ARA and EPA tend to produce less EPA and more ARA whencultivated at higher temperatures. For example, when Mortierella alpinais cultivated at less than 18° C., if begins to produce EPA. Thus, it ispreferable to maintain the temperature at a level which induces thepreferential production of ARA. Suitable temperatures are typically fromabout 25 ° C. to about 30° C.

Preferably, cultivation continues until a desired biomass density isachieved. A desirable biomass is about 25 g/l of the organism. Such abiomass typically is attained within 48-72 hours after inoculation. Atthis time, the organisms typically contain about 5-40% complex lipids,i.e. oil, of which about 10-40% is ARA, and can be harvested.

Harvesting can be done by any suitable method such as, for example,filtration, centrifugation, or spray drying. Because of lower cost,filtration may be preferred.

After harvesting, the mycelial cake can be extracted. The mycelial cakerefers to the collection of biomass resulting after harvest. The cakecan be loose or pressed, crumbled or uncrumbled. Optionally, the cakecan have any residual water removed, as by vacuum drying orlyophilization, prior to extraction. If this option is selected, it ispreferable to use nonpolar solvents to extract the ARA-containing oil.While any non-polar extract is suitable, hexane is preferred.

Alternatively, the wet cake (which typically contains about 30-50%solids) can be crumbled and extracted directly using polar solvents suchas ethanol or isopropyl alcohol, or supercritical fluid extraction withsolvents such as CO₂ or NO. Preferably, the cakes are crumbled prior toextraction. Advantageously, the present invention permits the economicaluse of supercritical fluid extraction techniques. McHugh, et al.,Supercritical Fluid Extraction, Butterworth (1986). Such techniques areknown to those of skill in the art and include those presently applied,for example, to decaffeinate coffee beans. While the yields from bothwet and dry extractions are similar, wet extraction generally is a moreeconomical process.

Another aspect of the invention discloses a process for supplementing oraltering the composition of commercially available infant formula so asto provide them with a PUFA composition more nearly like that typicallycontained in human breast milk. "Typical" as used herein refers to theaverage amounts of PUFAs measured. One of the advantages of the presentinvention is that, if desired, a nursing mother choosing to switch toformula can have her breast milk analyzed for PUFA content. Then, anadditive for a commercially available formula which will supplycomparable amounts of PUFAs can be specifically designed. Long chainPUFA-containing microbial oils from at least two microorganisms can beobtained and blended together to provide the desired composition. Theblend then can be added to an infant formula. Preferably, an amount ofthe blend effective to provide an amount of the desired PUFAssubstantially similar to that found in human breast milk will beprovided.

Typically, human breast milk contain from about 0.5 to 0.6% of its fattyacid content as ARA, from about 0.15 to about 0.36% of its fatty acidcontent as DHA and from about 0.03 to about 0.13% of its fatty acidcontent as EPA. Thus, a preferred ratio of ARA:DHA:EPA is from about5:1:1 to about 20:10:1 respectively. Amounts of oils providingapproximately these ratios of PUFAs can be determined without undueexperimentation by those of skill in the art.

In a preferred embodiment, the microbial oils include ARASCO and DHASCOand EPASCO or any combination thereof. It is also preferred to use oilfrom microbes of the genera Mortierella, Pythium, Crypthecodinium, andNitzschia or any combination thereof. Particularly preferred speciesfrom these genera are M. alpina, P. insidiosum, C. cohnii and N. alba.This preferred embodiment would provide an acceptable alternative forvegetarians, including breast-feeding or pregnant vegetarian women.

If desired, fish oil can be blended, or mixed, with any combination of,or individual, microbial oil to produce a composition which, whensubsequently added to infant formula will alter the PUFA content thereofin a desirable manner. Such a composition would not be suitable for astrict vegetarian intake. A preferred fish oil is specially processedMenhaden Oil (produced by Zapata Hayne, Inc.) which typically containsabout 9% DHA. Of course, other fish oils also can be used.

When DHASCO is to be blended with ARASCO, and no other PUFA-containingoils are to be utilized, it is desirable to blend sufficient amounts ofthe oils to provide from about 1 to about 5 parts DHA with from about 2to about 12 parts ARA. A most preferred ratio of DHA to ARA is 1:3respectively.

As another example, Menhaden fish oil, as noted above, typicallycontains about 9% by weight DHA. ARASCO typically contains about 20-40%by weight ARA. DHASCO typically contains about 25-40% by weight DHA. Ithas been found that a blend of 1 part Menhaden oil containing about 9%by weight DHA with 10 parts ARASCO containing about 33% by weight ARAand 3 parts DHASCO containing about 35% by weight DHA, when added toinfant formula, causes the infant formula to closely approximate the ARAand DHA content of human breast milk. Other ratios can be readilycalculated.

In another embodiment of the present invention is disclosed a processfor making a supplement for infant formula or baby food which entailsblending a DHA- containing oil with a GLA-containing oil. It is to beunderstood that, in general, any combination of GLA- EPA- ARA- orDHA-containing oils, with or without fish oil, can be used. The sourceof the GLA can be a vegetable oil, such as primrose, black currant orborage oil, or a microbial oil such as the oil from Mucor javonicus orMortierella isabellina, for example. Table 1 sets forth the GLAcomposition of such oils. In a preferred aspect of this embodiment,about 1 part of Menhaden oil containing about 9% DHA, about 4 parts ofGLA-containing oil containing about 18% GLA from black currant seed, andabout 1 part of DHASCO containing about 33% DHA are blended together.Other ratios can be selected as desired.

                  TABLE 1                                                         ______________________________________                                        Fatty acids of commercially available oils containing GLA                     (from Lawson and Hughes, 1988 and Suzuki, 1989)                               Relative % of total acyl groups in oil from:                                  Fatty acyl                                                                            Mucor     Mortierella                                                                             Evening                                                                              Black-                                     group   javanicus*                                                                              isabellina**                                                                            Primrose                                                                             currant                                                                             Borage                               ______________________________________                                        14:0    1.0       0.7       --     --    --                                   14:1    0.1       --        --     --    --                                   16:0    18.6      27.2      5.9    6.9   10.7                                 16:1    1.0       0.9       --     --    --                                   18:0    7.1       5.7       1.8    1.3   3.0                                  18:1    39.9      43.9      7.5    10.8  15.4                                 18:2    8.9       12.0      74.8   46.7  38.1                                 -18:3(6)                                                                              17.9      8.3       9.3    15.9  24.8                                 α-18:3(3)                                                                       --        --        --     13.0  --                                   18:4(3) --        --        --     2.9   --                                   20:0    --        0.6       --     --    --                                   20:1(9) --        --        --     --    4.0                                  22:0    --        0.1       --     --    --                                   22:1(9) --        0.2       --     --    2.2                                  24:0    0.6       --        --     --    --                                   ______________________________________                                         *Produced by J. & E. Sturge Ltd., Selby, N. Yorks., U.K.                      **Produced by Idemitzu Petro Chemical Co. Ltd., Tokyo, Japan.                 Lawson  Lipids 23:313-317 (1988)                                              Suzuki  In Biotechnology for the Fats and Oils Industry p.110-116. Amer       Oil Chem. Soc. Press (1989).                                             

A composition including a blend of any combination of theabove-described microbial-oils with or without either, or both, fish oiland vegetable oil is another aspect of the present invention. While thecomposition includes any ratios of the oils, the ratios previouslydescribed are preferred.

In another preferred embodiment, the composition serves as a nutritionalsupplement. Typically, such supplements are encapsulated, such as ingelatin capsules. Such capsules provide an easy form of administrationto persons having a need for supplementation, such as pregnant ornursing women. However, parenteral administration is a viable option andin one embodiment the composition comprises the fat component of a totalparenteral nutritional formula. Such formulas are known and commerciallyavailable.

As will be understood, the composition of the present invention isparticularly useful as a dietary supplement for pregnant or nursingwomen. Vegetarian women, in particular, may require increased amounts ofDHA and ARA, yet have been precluded from obtaining such in the pastbecause the only available sources were animal.

The invention having been previously described in general, reference isnow had to the following nonlimiting examples for illustrative purposesonly.

EXAMPLES Example 1

Preparation of Oil Mix #1 and addition to infant formula.

The first mixture represents a totally vegetarian source of anarachidonic and docosahexaenoic acid supplement. This supplement wouldbe considered acceptable to persons restricted to a vegetarian diet.Sanders et al. (Amer. J. Clin. Nutr. 31:705; 1978) have reported thatthe DHA levels in the breast milk of vegetarian mothers are depressed.Enteral supplementation of a blend of DHA single cell oil and ARA singlecell oil will elevate the serum and, hence, breast milk levels of DHA tothat of omnivorous mothers. This blend is prepared by mixing one partDHASCO containing about 35% DHA (obtained from Crypthecodinium cohnii asdescribed in U.S. Pat. No. 5,407,957 and deposited with the AmericanType Culture Collection in Rockville, Maryland (ATCC) having accessionnumber 40750) with three parts ARASCO containing about 33% ARA (obtainedfrom Pythium insidiosum as described in U.S. application Ser. No.07/645,454, now abandoned and on deposit with the ATCC, having accessionnumber 28251). The resulting mixture, or blend, has the fatty acidcomposition shown in Table 2. The blend is mixed in a ratio of one partblend to forty parts of the oils regularly in infant formula, typicallyabout 2.8-3.0 grams per 100 ml of formula. At a normal fat content of30g fat per liter of Similac® infant formula, this corresponds to theaddition of 750 mg per liter of prepared formula. This supplementprovides ARA and DHA levels equivalent to human breast milk.

                  TABLE 2                                                         ______________________________________                                        Composition of a blend of DHA oil and ARA oil in                              proportions of 1:3 by weight                                                                    Infant               breast                                 Fatty Acid                                                                            oil mix #1                                                                              formula  formula + mix #1                                                                          milk                                   ______________________________________                                        8:0 + 10:0                                                                            0.00      41.8     40.78       1.74                                   12:0 + 14:0                                                                           13.63     20.7     20.53       14.95                                  16:0    17.05     6.8      7.05        19.82                                  16:1    7.88      0.2      0.39        3.20                                   18:0    0.00      2.3      2.24        5.91                                   18:1    7.48      10.0     9.94        34.82                                  18:2 n6 7.20      17.4     17.15       16.00                                  18:3 n3 2.25      0.9      0.93        0.62                                   18:3 n6 4.50      --       0.11        0.00                                   20:1    --        0.1      0.10        1.10                                   20:2 n6 --        --       0.00        0.61                                   20:3 n6 --        --       0.00        0.42                                   20:4 n6 24.75     --       0.60        0.59                                   20:5 n3 --        --       0.00        0.03                                   22:1    --        --       0.00        0.10                                   22:4 n6 --        --       0.00        0.21                                   22:5 n6 --        --       0.00        0.22                                   22:6 n3 8.98      --       0.22        0.19                                   ______________________________________                                    

Example 2

Preparation of Oil Mix #2 and addition to infant formula.

This mixture represents a totally vegetarian source of long chain PUFAsand would be considered acceptable to persons restricted to a vegetariandiet. This blend is prepared by mixing three parts DHASCO containingabout 35% DHA (obtained from Crypthecodinium cohnii as described in U.S.Pat. No. 5,407,957 with ten parts ARASCO containing about 33% ARA(obtained from Pythium insidiosum as described in U.S. application Ser.No. 07/645,454 filed concurrently herewith) and five parts EPASCOcontaining about 5% EPA (obtained from N. alba on deposit with the ATCCas described in U.S. Pat. No. 5,244,921. The resulting mixture, orblend, has the fatty acid composition shown in Table 3. The blend ismixed in a ratio of one part blend to thirty parts of the oils regularlyin infant formula. At a normal fat content of 30g fat per liter ofSimilac® infant formula, this would correspond to the addition of onegram per liter of prepared formula. This supplement provides ARA, DHAand EPA levels equivalent to human breast milk.

                  TABLE 3                                                         ______________________________________                                        Composition of a blend of DHA oil, ARA oil and EPA oil                        in proportions of 3:10:5 by weight                                                              Infant               breast                                 Fatty Acid                                                                            oil mix #2                                                                              formula  formula + mix #2                                                                          milk                                   ______________________________________                                        8:0 + 10:0                                                                            0.00      41.8     40.45       1.74                                   12:0 + 14:0                                                                           16.64     20.7     20.57       14.95                                  16:0    21.61     6.8      7.28        19.82                                  16:1    6.55      0.2      0.40        3.20                                   18:0    0.28      2.3      2.23        5.91                                   18:1    12.91     10.0     10.09       34.82                                  18:2 n6 5.87      17.4     17.03       16.00                                  18:3 n3 1.88      0.9      0.93        0.62                                   18:3 n6 3.48      --       0.11        0.00                                   20:1    --        0.1      0.10        1.10                                   20:2 n6 --        --       0.00        0.61                                   20:3 n6 0.19      --       0.01        0.42                                   20:4 n6 18.52     --       0.60        0.59                                   20:5 n3 0.76      --       0.02        0.03                                   22:1    --        --       0.00        0.10                                   22:4 n6 0.11      --       0.00        0.21                                   22:5 n6 --        --       0.00        0.22                                   22:6 n3 6.24      --       0.20        0.19                                   ______________________________________                                    

Example 3

Preparation of Oil Mix #3 and addition to infant formula.

This mixture is a blend of ARASCO with fish oils. Oil mixture #3 isprepared by adding one part specially processed Menhaden Oil (ZapataHayne Inc.) containing about 9% DHA to one part of ARASCO, obtained fromPythium insidiosum as described previously containing about 33% ARA. Theresultant fatty acid composition is shown in Table 4. This blend ismixed in a ratio of one part blend to thirty parts of the oils regularlyin infant formula. At a normal fat content of 30 g fat per liter ofinfant formula, this corresponds to the addition of 1 gram per liter ofprepared formula. This supplement provides ARA and DHA levels equivalentto human breast milk, but the EPA levels are about eight-fold higherthan those in breast milk.

                  TABLE 4                                                         ______________________________________                                        Composition of a blend of SPMO* and ARA oil in                                proportions of 1:1 by weight.                                                                   Infant               breast                                 Fatty Acid                                                                            oil mix #3                                                                              formula  formula + mix #3                                                                          milk                                   ______________________________________                                        8:0 + 10:0                                                                            0.0       41.8     40.45       1.74                                   12:0 + 14:0                                                                           10.2      20.7     20.36       14.95                                  16:0    15.5      6.8      7.08        19.80                                  16:1    11.5      0.2      0.56        3.20                                   18:0    1.41      2.3      2.27        5.91                                   18:1    8.79      10.0     9.96        34.82                                  18:2 n6 5.57      17.4     17.02       16.00                                  18:3 n3 2.31      0.9      0.95        0.62                                   18:3 n6 3.00      --       0.10        0.00                                   20:1    0.78      0.1      0.12        1.10                                   20:2 n6 0.00      --       0.00        0.61                                   20:3 n6 0.00      --       0.00        0.42                                   20:4 n6 17.52     --       0.57        0.59                                   20:5 n3 7.76      --       0.25        0.03                                   22:1    0.00      --       0.00        0.10                                   22:4 n6 0.00      --       0.00        0.21                                   22:5 n6 1.21      --       0.04        0.22                                   22:6 n3 4.57      --       0.15        0.19                                   ______________________________________                                         * Specially Processed Menhaden Oil.                                      

Example 4

Preparation of Oil Mix #4 and addition to infant formula

Oil mixture #4 was developed to utilize GLA in place of arachidonicacid. This blend was prepared by mixing one part specially preparedMenhaden oil containing about 9% DHA (Zapata Hayne Inc.) with four partsblack currant seed oil containing about 18% GLA and one part DHASCOcontaining about 35% DHA. The resultant fatty acid composition is shownin Table 5. This blend is mixed in a ratio of one part blend to fortyparts of the oils regularly in infant formula. At a normal fat contentof 30g fat per liter, this would correspond to the addition of 750 mgper liter of prepared formula. This supplement provides EPA and DHAlevels equivalent to human breast milk. The ARA levels are about onetenth the level in human breast milk. However, the GLA levels are twentyto fifty times higher than the GLA levels in breast milk which typicallyare minute.

                  TABLE 5                                                         ______________________________________                                        Composition of a blend of SPMO, BCO and DHA oil in                            proportions of 1:4:1 by weight.                                                                 Infant               breast                                 Fatty Acid                                                                            oil mix #4                                                                              formula  formula + mix #4                                                                          milk                                   ______________________________________                                        8:0 + 10:0                                                                            0.0       41.8     40.78       1.74                                   12:0 + 14:0                                                                           4.83      20.7     20.31       14.95                                  16:0    11.86     6.8      6.92        19.80                                  16:1    2.09      0.2      0.25        3.20                                   18:0    1.34      2.3      2.28        5.91                                   18:1    10.98     10.0     10.02       34.82                                  18:2 n6 31.39     17.4     17.74       16.00                                  18:3 n3 8.94      0.9      1.10        0.62                                   18:3 n6 10.60     --       0.26        0.00                                   20:1    0.26      0.1      0.10        1.10                                   20:2 n6 --        --       0.00        0.61                                   20:3 n6 --        --       0.00        0.42                                   20:4 n6 0.34      --       0.06        0.59                                   20:5 n3 2.59      --       0.00        0.03                                   22:1    --        --       0.00        0.10                                   22:4 n6 --        --       0.00        0.21                                   22:5 n6 0.40      --       0.01        0.22                                   22:6 n3 7.51      --       0.18        0.19                                   ______________________________________                                    

Example 5

Preparation of Oil Mix #5 and addition to infant formula

Oil mixture #5 was developed to best approximate the composition of DHA,ARA and EPA of human breast milk. This oil blend was prepared by mixingone part specially prepared Menhaden oil containing about 9% DHA (ZapataHayne Inc.) with ten parts of ARASCO containing about 33% ARA and threeparts DHASCO containing about 35% DHA. The resultant fatty acidcomposition is shown in Table 6. This blend is mixed in a ratio of onepart blend to forty parts of the oils regularly in infant formula. At anormal fat content of 30g fat per liter of infant formula, thiscorresponds to the addition of 750 mg per liter of prepared formula.This supplement provides EPA, DHA and ARA levels substantiallyequivalent to those levels in human breast milk.

                  TABLE 6                                                         ______________________________________                                        Composition of a blend of SPMO, ARA oil and DHA oil                           in proportions of 1:10:3 by weight.                                                             Infant               breast                                 Fatty Acid                                                                            oil mix #5                                                                              formula  formula + mix #5                                                                          milk                                   ______________________________________                                        8:0 + 10:0                                                                            0.00      41.8     40.78       1.74                                   12:0 + 14:0                                                                           13.14     20.7     20.52       14.95                                  16:0    16.83     6.8      7.04        19.80                                  16:1    8.39      0.2      0.40        3.20                                   18:0    0.20      2.3      2.25        5.91                                   18:1    7.66      10.0     9.94        34.82                                  18:2 n6 6.97      17.4     17.15       16.00                                  18:3 n3 2.26      0.9      0.93        0.62                                   18:3 n6 0.25      --       0.01        0.00                                   20:1    0.11      0.1      0.10        1.10                                   20:2 n6 --        --       0.00        0.61                                   20:3 n6 --        --       0.00        0.42                                   20:4 n6 23.72     --       0.58        0.59                                   20:5 n3 1.11      --       0.03        0.03                                   22:1    --        --       0.00        0.10                                   22:4 n6 --        --       0.00        0.21                                   22:5 n6 0.17      --       0.00        0.22                                   22:6 n3 8.35      --       0.20        0.19                                   ______________________________________                                    

We claim:
 1. A process for making a supplement for infant formulaconsisting essentially of blending a docosahexaenoic acid(DHA)-containing microbial oil and a gamma linolenic acid(GLA)-containing oil, and adding an amount of the blended oils to infantformula, such that the amount of DHA provided by the blend is sufficientto provide an amount of DHA comparable to the amount in human breastmilk and the amount of GLA provided by the blend is sufficient toprovide GLA in an amount that, upon administration of the formula to aninfant, can be converted in the infant's body to an amount ofarachidonic acid (ARA) comparable to the amount of ARA obtainable fromhuman breast milk.
 2. The process of claim 1, wherein said linolenicacid-containing oil comprises primrose, borage, or black currant seedoil.
 3. The process of claim 1, wherein said linolenic acidcontaining-oil comprises a microbial oil.
 4. The process of claim 3,further comprising obtaining said linolenic acid-containing oil fromMucor javonicus or Mortierella isabellina.
 5. The process of claim 1,further comprising blending an EPA-containing oil with saidDHA-containing microbial oil and said linolenic acid-containing oil. 6.The process of claim 5, wherein said EPA-containing oil comprises fishoil.
 7. The process of claim 6, wherein said fish oil comprises aboutone part, said linolenic acid-containing oil comprises about 4 parts andsaid DHA-containing oil comprises about 1 part by weight of said blend.8. The process of claim 6, wherein said fish oil comprises about onepart, said linolenic acid-containing oil comprises about 4 parts andsaid DHA-containing oil comprises about 1 part by weight of said blend.9. A composition consisting essentially of a blend of a DHA-containingmicrobial oil and a gamma linolenic acid-containing oil, in a ratio suchthat adding to infant formula an amount of the blended oil compositionsufficient to provide an amount of the DHA comparable to the amount inhuman breast milk will provide an amount of GLA in the formula that,upon administration of the formula to an infant, can be converted in theinfant's body to an amount of ARA comparable to the amount Of ARAobtainable from human breast milk.
 10. The composition of claim 9,wherein said linolenic acid-containing oil comprises primrose, borage,or black currant seed oil.
 11. The composition of claim 9, wherein saidlinolenic acid containing-oil is an oil obtained from a microbe.
 12. Thecomposition of claim 11, wherein said microbe comprises Mucor javonicusor Mortierella isabellina.
 13. The composition of claim 9, alsocomprising an EPA-containing oil, wherein the amount of EPA provided bythe blend is less than or equal to one-fifth of the amount of ARAobtained from conversion of GLA in the infant's body.
 14. Thecomposition of claim 13, wherein said EPA-containing oil comprises fishoil.
 15. The composition of claim 14, wherein said fish oil comprisesabout one part, said linolenic acid-containing oil comprises about 4parts and said DHA-containing oil comprises about 1 part by weight ofsaid blend.
 16. A composition comprising a blend of an oil containingDHA and an oil containing ARA, wherein DHA and ARA are in the form oftriglycerides and further wherein at least one of the oil containing DHAand the oil containing ARA is a microbial oil and ARA:EPA ratio of theblend is at least 5:1.
 17. A composition according to claim 16, whereinthe oils are blended to provide a ratio of about 2 to 12 parts ARA toabout 1 to 5 parts DHA.
 18. A composition in accordance with claim 17,wherein the microbial oil containing DHA comprises about 25-40% DHA. 19.A composition in accordance with claim 17, wherein the microbial oilcontaining DHA comprises at least about 35% DHA.
 20. A composition inaccordance with claim 17, wherein the microbial oil containing ARAcomprises about 20-40% ARA.
 21. A composition in accordance with claim17, wherein the oils are blended to provide a ratio of ARA:DHA of about2:1.
 22. The composition according to any one of claims 9 or 17,comprising fish oil blended with at least one microbial oil.
 23. Thecomposition of claim 22, wherein the ratio of fish oil to microbial oilis from 1:1 to 1:15.
 24. A composition according to claim 16, whereinthe oils are blended to provide an ARA:DHA:EPA ratio of from about 5parts ARA and 1 part DHA to about 20 parts ARA and 10 parts DHA to about1 part EPA.
 25. A composition according to claim 16, comprising a blendof a microbial triglyceride oil containing ARA, and a microbialtriglyceride oil containing DHA, wherein the microbial triglyceride oilsare blended in amounts to provide a ratio of about 2 to 12 parts ARA toabout 1 to 5 parts DHA and said oils further are essentially free ofEPA.
 26. A nutritional supplement containing the composition of any oneof claims 9, 16 or
 17. 27. A nutritional supplement according to claim26, comprising a blend of a microbial oil containing DHA and a microbialoil containing ARA, wherein the oils are blended to provide a ratio ofabout 2 to 12 parts ARA and about 1 to 5 parts DHA, said oils having ARAand DHA in the form of triglycerides.
 28. The nutritional supplementaccording to claim 26, wherein the supplement is a human nutritionalsupplement.
 29. The nutritional supplement according to claim 26,wherein the human is a baby.
 30. The nutritional supplement according toclaim 26, wherein the human is a pregnant or nursing woman.
 31. A totalparenteral nutrition formula containing the composition according to anyof claims 9, 16, or 17.